Cell chemotaxis assays

ABSTRACT

A device includes an input chamber, an attractant chamber, a migration channel arranged in fluid communication between an outlet of the input chamber and inlet of the attractant chamber, a baffle arranged in fluid communication between the outlet of the input chamber and the migration channel or within the migration channel, and an exit channel in fluid communication with the migration channel at a point beyond the baffle and before the migration channel enters the inlet of the attractant chamber. The baffle is configured to inhibit movement of a first type of cell through the baffle to a greater extent than the baffle inhibits movement of a second type of cell through the baffle.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national stage application under 35 USC § 371of International Application No. PCT/US2014/056614, filed on Sep. 19,2014, which claims priority to U.S. Provisional Application No.61/880,591, filed on Sep. 20, 2013, the entire contents of which areincorporated herein by reference.

TECHNICAL FIELD

The present disclosure relates to cell chemotaxis assays.

BACKGROUND

Neutrophil directional migration in response to chemical gradients, alsoknown as chemotaxis, is one of the key phenomena in immune responsesagainst bacterial infection and tissue injury. Alterations in neutrophilchemotaxis, e.g., as a result of burns or trauma, may lead to chronicinflammation and further tissue damage. Identifying alterations ofneutrophil chemotaxis may help estimate the risk for infections moreaccurately. To better study neutrophil chemotaxis, in vitro assays havebeen developed that replicate chemotactic gradients around neutrophils.Since red blood cells out-number neutrophils and have the propensity toclot, measurements of neutrophil migration pattern in whole blood ischallenging. For this reason, traditional assays (e.g. transwells) usesisolated neutrophil cells from whole blood.

However, existing assays can include one or more drawbacks. For example,such assays require time-consuming processing of blood to isolateneutrophils. Furthermore, such isolation may alter the responsiveness ofneutrophils compared to in vivo conditions, leading to inaccuratecharacterization of the neutrophils. For instance, certain chemotaxisassays utilize cell separation methods, such as positive selection ornegative selection, which are prone to activating neutrophils byengaging specific receptors on the neutrophils. Once activated, theneutrophils' migration profile can be altered; however, this change maynot be directly related to the biological condition of interest, butrather a response from the applied stress introduced by cell isolationprotocols. Additionally, existing assays require processing relativelylarge volumes of blood. These assays lack accuracy and/or require use bytechnicians having specialized training. These limitations, amongothers, restrict the assays' usefulness in clinical laboratories.

SUMMARY

The microfluidic devices disclosed herein enable the investigation ofcell motility, such as neutrophil chemotaxis in blood samples. Inparticular, the microfluidic devices can be used to measure thedirectional migration and speed of neutrophils in an attractantgradient, using low sample volumes and with minimal, if any, activationof the neutrophils. Using the new devices, an analysis of cell motilityhealth or impairment, e.g., due to infection or tissue injury, can bedetermined with a high degree of precision. Moreover, the new devicescan be used to identify candidate drug agents suitable for modifyingcell motility. Such compounds then can be further screened for theirpotential use as mediators of inflammation resulting from tissue traumaand/or infections.

In particular, the new devices circumvent the need to use separationmethods that interfere with the motile cell analysis (e.g., positive ornegative selection) by relying instead on a baffle structure thatinhibits the movement of undesired cells in a sample to a greater extentthan the movement of the desired motile cells. For neutrophil analysis,the new devices allow whole blood samples to be used directly, and thusreduce the overall sample processing time, while also enabling analysisof the neutrophils directly without the need to isolate them from wholeblood.

In general, the subject matter disclosed herein can be embodied inmethods for monitoring neutrophil chemotaxis in a device, in which themethods include obtaining a device that includes an input chamber, anattractant chamber, a migration channel arranged in fluid communicationbetween an outlet of the input chamber and an inlet of the attractantchamber, a baffle arranged in fluid communication between the outlet ofthe input chamber and the migration channel or within the migrationchannel, and an exit channel in fluid communication with the migrationchannel at a point beyond the baffle and before the migration channelenters the inlet of the attractant chamber. The methods further includeadding an attractant solution to the device to establish an attractantgradient between the input chamber and the attractant chamber, adding tothe input chamber a blood sample including a plurality of red bloodcells and a plurality of neutrophils, incubating the device underconditions and for a time sufficient to enable movement of cells in theblood sample from the input chamber into the migration channel, in whichthe baffle is configured to inhibit movement of the red blood cellsthrough the baffle to a greater extent than the baffle inhibits movementof the neutrophils through the baffle, and monitoring whether any of theneutrophils follow the attractant gradient in the migration channeltoward the attractant chamber.

The methods can include one or more of the following features in variouscombinations. For example, in some implementations, the blood sample iswhole blood.

In some implementations, monitoring whether any of the neutrophilsfollow the attractant gradient includes determining a number ofneutrophils that follow the attractant gradient.

In certain instances, the blood sample has a volume less than about 2microliters.

In some cases, establishing the attractant gradient includes adding theattractant solution to all chambers and channels in the device, andreplacing the attractant solution in the input chamber with a liquidmedium that lacks the attractant such that the attractant gradient formsbetween the input chamber and the attractant chamber.

In some implementations, the attractant solution includes interleukin-8(IL-8), C5a, formyl-methionyl-leucyl-phenylalanine (fMLP), leukotrieneB₄ (LTB₄), adhenosine tri-phosphate (ATP), tumor growth factor beta(TGFb), or endothelial derived neutrophil attractant factor (ENA).

In some instances, the baffle includes a first fluid passage in fluidcommunication with a second fluid passage, in which an angle between asample transport path in the first fluid passage and a sample transportpath the second fluid passage is greater than or equal to about 45degrees. For example, the angle can be about 90 degrees.

In some implementations, a cross-sectional area of the first fluidpassage normal to the sample transport path in the first fluid passageis greater than a red blood cell cross-sectional area, a height of thecross-sectional area is greater than a red blood cell thickness and lessthan a red blood cell diameter, and a width of the cross-sectional areais greater than the red blood cell diameter. For example, the height ofthe cross-sectional area can be greater than about 2 microns and lessthan about 6 microns, and the width of the cross-sectional area can begreater than about 6 microns.

In certain implementations, the baffle includes multiple first fluidpassages, and multiple second fluid passages, each first fluid passagebeing in fluid communication with the output of the input chamber and influid communication with a corresponding second fluid passage, in whichan angle between a fluid transport path of each first fluid passage anda sample transport path of the corresponding second fluid passage isgreater than or equal to about 45 degrees.

In some implementations, the methods further include analyzing thehealth of the neutrophils that follow the attractant gradient in themigration channel toward the attractant chamber. Analyzing the health ofthe neutrophils can include determining a number of the neutrophils thatfollow the attractant gradient toward the attractant chamber compared toa total number of cells moving through the migration channel,determining a rate at which one or more neutrophils follow theattractant gradient toward the attractant chamber, and/or determining anumber of neutrophils that do not follow the attractant gradientcompared to the total number of moving cells.

In some cases, monitoring whether any of the neutrophils follow theattractant gradient includes obtaining an image of neutrophils in theattractant chamber. Monitoring whether any of the neutrophils follow theattractant gradient can occur for at least 10 minutes.

In some implementations, adding the attractant solution to establish theattractant gradient includes applying a vacuum to the input chamber, themigration channel, the baffle and the attractant chamber such that airis absorbed through walls of the device.

In another aspect, the subject matter of the disclosure can be embodiedin methods of screening neutrophil attractants, in which the methodsinclude obtaining a device that includes an input chamber, multipleattractant chambers, and multiple filtration passageways, eachfiltration passageway being in fluid communication with the inputchamber and a corresponding attractant chamber, and each filtrationchamber including a migration channel in fluid communication between anoutlet of the input chamber and an inlet of the corresponding attractantchamber, a baffle arranged in fluid communication between the outlet ofthe input chamber and the migration channel or within the migrationchannel, and an exit channel in fluid communication with the migrationchannel at a point beyond the baffle and before the migration channelenters the inlet of the corresponding attractant chamber. The methodsfurther includes adding a different attractant solution to at least twoattractant chambers to establish a different attractant gradient betweenthe input chamber and each attractant chamber to which an attractantsolution has been added, adding to the input chamber a blood sampleincluding multiple red blood cells and multiple neutrophils, incubatingthe device under conditions and for a time sufficient to enable movementof cells in the blood sample from the input chamber into one or morefiltration passageways, in which the baffles of the one or morefiltration passageways are configured to inhibit movement of the redblood cells through the baffles to a greater extent than the bafflesinhibit movement of the neutrophils through the baffles, and monitoringwhether any of the neutrophils follow any of the established attractantgradients to one of the attractant chambers to which an attractantsolution has been added.

The methods can include one or more of the following features in variouscombinations. For example, in some implementations, monitoring whetherany of the neutrophils follow any of the established attractantgradients includes, for each different attractant gradient, determininga number of neutrophils that follow the attractant gradient and/ordetermining a rate at which one or more neutrophils follow theattractant gradient.

In some implementations, the methods further include identifying theattractant that establishes the attractant gradient resulting in thelargest number of neutrophils reaching an attractant chamber and/orresulting in the highest rate at which one or more neutrophils followthe attractant gradient. Each baffle can include multiple first fluidpassages being in fluid communication with the output of the inputchamber, and multiple second fluid passages, each second fluid passagebeing in fluid communication with a corresponding first fluid passage ofthe baffle, in which an angle between a fluid transport path of eachsecond fluid passage and a sample transport path of the correspondingfirst fluid passage is greater than or equal to about 45 degrees.

In some cases, a cross-sectional area of each first fluid passage isgreater than a red blood cell cross-sectional area, a height of thecross-sectional area for each first fluid passage is greater than a redblood cell thickness and less than a red blood cell diameter, and awidth of the cross-sectional area is greater than the red blood celldiameter.

In another aspect, the subject matter of the present disclosure can beembodied in a device or devices that include an input chamber, anattractant chamber, a migration channel arranged in fluid communicationbetween an outlet of the input chamber and inlet of the attractantchamber, a baffle arranged in fluid communication between the outlet ofthe input chamber and the migration channel or within the migrationchannel, and an exit channel in fluid communication with the migrationchannel at a point beyond the baffle and before the migration channelenters the inlet of the attractant chamber. The baffle is configured toinhibit movement of a first type of cell through the baffle to a greaterextent than the baffle inhibits movement of a second type of cellthrough the baffle.

The device or devices may include one or more of the following featuresin various combinations. For example, in some implementations, thebaffle includes a first fluid passage in fluid communication with asecond fluid passage, in which an angle between a sample transport pathin the first fluid passage and a sample transport path the second fluidpassage is greater than or equal to about 45 degrees. For example, theangle can be about 90 degrees.

In some implementations, a cross-sectional area of the first fluidpassage normal to the sample transport path in the first fluid passageis greater than a cross-sectional area of the first type of cell, aheight of the cross-sectional area is greater than a thickness of thefirst type of cell and less than a diameter of the first type of cell,and a width of the cross-sectional area is greater than the diameter ofthe first type of cell. The height of the cross-sectional area can begreater than about 2 microns and less than about 6 microns, and whereinthe width of the cross-sectional area can be greater than about 6microns.

In some cases, a volume of the input chamber is greater than about 1microliter and less than about 5 microliters.

In some implementations, a length of a fluid transport path through themigration channel is between about 10 microns and about 2000 microns.

In certain cases, the baffle multiple first fluid passages, and multiplesecond fluid passages, each first fluid passage being in fluidcommunication with the output of the input chamber and in fluidcommunication with a corresponding second fluid passage, in which anangle between a fluid transport path of each first fluid passage and asample transport path of the corresponding second fluid passage isgreater than or equal to about 45 degrees.

In some implementations, the inlet chamber and/or the migration channelis coated with a protein. In some cases, the protein is configured toprevent neutrophil adhesion. The protein can be albumin. In some cases,the protein is configured to promote neutrophil activation. The proteincan be P selectin.

In another aspect, the subject matter of the present disclosure can beembodied in a system that includes any of the foregoing devices and acontrol apparatus configured to image a number of cells migrating fromthe input chamber through the baffle and the migration channel to theattractant chamber

As used herein, “motility” means the ability of a motile cell to moveitself, e.g., at a specific migration rate, at least under certainconditions. Motile cells include neutrophils and other immune cells suchas granulocyte, monocytes, and lymphocytes, as well as certain cellsthat can move only under certain specific conditions, such as mast cellprecursors, fibroblasts, and endothelial cells (e.g., circulatingendothelial cells (“CEC”)) or cells under pathological conditions, suchas metastatic cancer cells (e.g., circulating tumor cells (“CTC”)).Other examples of motile cells include, but are not limited to, spermcells, bacteria, parasites (e.g., sporozoite phase parasites),eosinophil cells, dendritic cells, and platelets.

As used herein, “chemotaxis” means a movement of a motile cell inresponse to a chemical stimulus.

As used herein, “attractant” means an agent or force that induces amotile cell to migrate towards the agent. Any agent that activatesmigration may be used as an attractant, including, for example, agentsthat introduce a force that is chemically, mechanically, or electricallybased.

As used herein, “repellant” means an agent that induces a motile cell tomigrate away from the agent. Any agent that activates migration may beused as a repellant, including, for example, agents that introduce aforce that is chemically, mechanically, or electrically based.

As used herein, “gas-permeable” means having openings that allow gas topass through.

As used herein, “blood sample” means any treated or non-treated blood.

Implementations of the subject matter described herein can includeseveral advantages. For example, in some instances, the presentlydescribed techniques bypass the need to purify/isolate motile cells froma fluid sample, such as neutrophils from whole blood, prior toperforming an assay. In certain implementations, the microfluidicdevices disclosed herein enable identification of motile cells, such asneutrophils, without requiring the use of cumbersome cell separationmethods such as density gradients, positive selection, or negativeselection, which can introduce artifacts by activating neutrophils. Thepresently disclosed methods do not require a washing step to wash bloodfrom the device after neutrophils are identified. Since such washingsteps typically require additional hardware external to the microfluidicdevice, cost and complexity can be reduced. Neutrophil migration in thepresently disclosed devices takes place inside three-dimensionalchannels, as opposed to solely on a two-dimensional surface, which mayinduce changes in speed and direction. Thus, the present device enablesaccurate quantification of neutrophil migration. In some cases, usefulquantitative data can be obtained just by counting motile cells thatreach an attractant chamber at the end of the assay, as opposed torequiring cell tracking.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1A is a schematic example of a microfluidic device as describedherein.

FIG. 1B is a close-up view of a portion of the device of FIG. 1A.

FIG. 2 is a schematic depicting an example of a baffle passagewaycross-section.

FIGS. 3A-3D depict stages of microfluidic assay preparation.

FIG. 4 is a schematic of an example of a system for analyzing an assayperformed in a microfluidic device.

FIGS. 5A-5D are schematics depicting the different stages of red bloodcells moving through a channel from an input chamber of a microfluidicdevice and subsequently traversing a 90° turn.

FIG. 6A is a bright-field (BF) image of a microfluidic device without abaffle to filter red blood cells.

FIG. 6B is a BF image of a microfluidic device fabricated with a baffleto filter red blood cells.

FIGS. 7A-7H are time-lapse images of a microfluidic device through whichred blood cells and neutrophils migrate.

FIG. 8A is a plot of neutrophil migration counts for neutrophils from avenous blood source, a finger prick blood source, and for isolatedneutrophils.

FIG. 8B is a plot of neutrophil migration counts per attractant chamberfor whole blood samples obtained from seven different subjects.

FIG. 8C is a plot of average neutrophil migration counts in six separatemicrofluidic devices.

FIG. 8D is a plot of baseline neutrophil migration in a healthy subjectover a three week time period.

FIG. 9A is a plot of the dose-response of neutrophils migrating out ofwhole blood to the leukotriene B4 (LTB₄) andN-formyl-methionyl-leucyl-pheylalanine (fMLP) chemoattractants.

FIG. 9B is a plot of neutrophil velocity in response to LTB₄ and fMLPchemoattractants for venous blood and finger prick blood.

FIG. 9C is a plot that shows a directionality index for neutrophils inresponse to both fMLP and LTB₄.

FIG. 9D is a plot that shows a neutrophil count in the fMLP attractantchamber for pin prick and venous blood in response to attractant withand without fibronectin.

FIG. 10A is a plot of neutrophil migration counts in an attractantchamber with no chemoattractant gradient, with fMLP chemoattractantgradient, and with LTB₄ chemoattractant gradient.

FIG. 10B is a plot of velocity of neutrophils migrating to LTB₄ comparedwith fMLP over a three week period.

FIG. 10C is a plot of a directionality index of neutrophils migrating toLTB₄ compared with fMLP.

FIG. 11A is a plot of the percentage of cells that have migrated to LTB₄compared with fMLP for human, rat, and mouse cell strains.

FIG. 11B is a plot of cell migration velocity for each different cellstrain.

FIG. 11C is a plot of directionality index for each different cellstrain.

FIG. 12A is a plot of the percentage of human and mouse cells that havemigrated in response to C5a.

FIG. 12B is a plot of the percentage of neutrophil cells activated inresponse to C5a.

FIG. 12C is a directionality plot of human and mouse cells towards C5a.

FIG. 12D is a directionality plot of human and mouse cells towards LBT₄.

DETAILED DESCRIPTION

FIG. 1A is a schematic example of a microfluidic device 100 foranalyzing cell motility, e.g., neutrophil motility. The microfluidicdevice 100 can be used for inducing migration of motile cells in anattractant gradient. The device 100 includes an input chamber 102 forreceiving a fluid sample, where the fluid sample may contain multiplemotile cells and non-motile cells. For example, the fluid sample couldbe a droplet of whole blood that contains both neutrophils and red bloodcells (RBCs). As shown in the example of FIG. 1A, the input chamber 102is surrounded by one or more attractant chambers 104, in which a motilecell attractant may be provided. Each of the attractant chambers 104 isfluidly coupled to the input chamber 102 through a baffle 106 and afluid migration channel 108. In the present example, the input chambers102 have a circular profile, though other shapes may also be used. Thediameter of the input chamber 102 can be between about 100 microns toabout 2000 microns. For example, the diameters can be about 200, 250,300, 350, 400, 450, 500, 750, or 1000 microns. If the diameter is toolarge, it will increase the time required for the motile cells, e.g.,neutrophils, far from the baffle 106 to exit the loading chamber andenter the next part of the device.

The input chamber 102 can have a volume in the range of about 1microliter to about 20 microliters. For example, the input chamber 102can have a volume of about 2, 3, 4, 5, 7, 8, 10, 12, 15, 18, or 20microliters. The one or more attractant chambers 104 have rectangularshaped profiles in the present embodiment, but other shapes can also beused. The volume of the attractant chambers 104 can be between about10-1000 nanoliter. For example, the volume of the attractant chamber canbe about 50 to 750 nanoliters, or 100 to 500 nanoliters.

FIG. 1B is a close-up view of a portion of the device of FIG. 1A,showing one of the attractant chambers 104 and the corresponding baffle106 and migration channel 108 to which the attractant chamber 104 iscoupled. As shown in FIG. 1B, the baffle 106 is arranged in fluidcommunication between an outlet of the input chamber 102 and themigration channel 108, such that a fluid sample deposited in the inputchamber can move through the baffle 106 into the migration channel 108.The migration channel 108 is arranged in fluid communication between anoutlet of the baffle 106 and an inlet of the attractant chamber.

During operation of the device 100, an attractant solution is added tothe attractant chamber 104 to establish an attractant concentrationgradient between the input chamber 102 and the attractant chamber 104.In general, the concentration of the attractant is highest in theattractant chamber 104 and decreases from the chamber 104 through themigration channel 108 and the baffle 106 to the input chamber 102. Whena fluid sample containing a motile cell responsive to the attractant isadded to the input chamber 102, the attractant concentration gradientinduces chemotaxis of the motile cell toward the region where theconcentration of the attractant is highest, i.e., the attractant chamber104. Both the baffle 106 and the migration channel 108 are sized toallow the desired motile cell to pass from the input chamber 102 to theattractant chamber 104.

After establishing the attractant gradient, the input chamber 102 isfilled with the fluid sample. Cells within the fluid sample begin movingtoward the openings of the baffle(s) 106. Cell movement does not occurbased on external pressure source (e.g., introducing pressuredifferences using syringe pumps or liquid pumps) or any flow of theliquid within the device. Instead, cell movement through the fluid inthe device is the result of a combination of passive factors including astatic pressure difference created by filling the input chamber with thefluid sample, natural diffusion, and/or random Brownian motion. Theprimary mechanism for motion of motile cells (e.g., by “crawling” in thecase of neutrophils or swimming for other motile cells such as certainbacteria and sperm) in the fluid sample will be in response to theexistence of an attractant gradient, depending on the choice of theattractant, e.g., chemoattractant.

As explained above, to help prevent the undesired cells from inhibitingor interfering with the active migration of the motile cells, the baffle106 is configured to inhibit the movement of undesired cells to agreater extent than the desired motile cells. For example, in the device100 shown in FIG. 1B, the baffle 106 includes one or more passageways110 sized to selectively allow migration of the desired motile cells,while being small enough to substantially block the movement of otherundesired motile and non-motile cells into the migration channel 108.For instance, for neutrophil analysis in a whole blood sample, thedimensions of the passageways 110 are sized to allow migration ofneutrophils in the blood sample, while being small enough tosubstantially impede the passage of other cells in the sample, such asRBCs and other leukocytes (e.g., monocytes and lymphocytes). Whileneutrophils are generally the same size or larger than RBCs and otherleukocytes (e.g., a typical human neutrophil is between about 8-15microns in diameter; typical human lymphocytes and monocytes havediameters of about 7 microns and between about 10-30 microns,respectively; a typical disc-shaped human RBC has a diameter betweenabout 6-8 microns and a height between about 2-2.5 microns), neutrophilsare more deformable in shape than RBCs and other blood cells, and thuscan change dimensions to migrate through tight passages that wouldotherwise impede the movement of other leukocytes and RBCs.

One way of appropriately sizing the passageways 110 to allowneutrophils, but not other cells to pass through is to restrict thecross-sectional area of the passageway along a plane normal to thedirection of cell movement. For example, one could use microfluidicchannels having cross sections smaller than that of the undesired cells.However, such channels could be completely obstructed at their entranceby a collection of cells, precluding the formation of gradients.Furthermore, cross-sectional areas that are smaller than that of theundesired cells could also impede the desired motile cell migration,since such cells would have no gaps to pass through. Instead, for themicrofluidic devices disclosed herein, the cross-sectional areas of thepassageways 110 and/or of the migration channels 108 are configured tobe larger than the largest diameter or cross-sectional dimension of oneor more of the different undesired cells. At the same time, a firstdimension of the passageway and/or migration channel cross-section isconfigured to be about equal to or less than a size of the undesiredcell. With this configuration, substantial movement of the undesiredcell(s) through the passageway 110 is still be restricted, but thedesired motile cell (e.g., neutrophil) modifiesits shape as it followsthe attractant gradient so as to migrate around and between theundesired cells through open gaps in the passageway 110. The desiredmotile cells thus “squeeze” their way around and between the undesiredcells. As an example, a first dimension of the cross-section (e.g.,width) can be set larger than the cell(s) to be blocked, whereas asecond dimension of the cross-section (e.g., height) is set to beshorter than the cell(s) to be blocked.

FIG. 2 is a schematic depicting an example of a passageway cross-sectionalong a plane that is normal to a direction of transport through thepassageway 110 (the direction of transport in this example is along they-direction into the page). The cross-section is bound by walls 200 ofthe passageway 110. For separating RBCs and undesired leukocytes fromneutrophils in a human blood sample, the height 202 of the passageway110 is configured to be less than the diameter of a RBC, e.g., betweenabout 1-6 microns. Additionally, the width 204 of the passageway 110 isconfigured to be larger than the diameter of the RBC, e.g., betweenabout 8-12 microns. With such dimensions, the device tends to force RBCstend to lay on their side before entering the passageway 110, thuslimiting RBC passage. Furthermore, monocytes and lymphocytes tend torequire much larger openings (e.g., at least 10 microns by 10 microns)to allow migration. However, given the relatively large passagewaywidth, there is still room for the neutrophils to migrate past the RBCsand other leukocytes. Furthermore, the larger width prevents completeclogging of the passageway 110 by the RBCs and other leukocytes. If thechannel cross-section is too small (e.g., less than 1 μm), gaps do notform such that the attractant gradient cannot be maintained and theneutrophils do not have enough room, even after deforming their shape,to pass through into the migration channel 108. The cross-sectional areaof the passageways 110 can be smaller or larger than those describedhere, and can be used to analyze the migration of cells other thanneutrophils including, for example, lymphocytes, monocytes, naturalkiller lymphocytes, platelets and megacariocytes, epithelial cells,endothelial cells, cancer cells, bacteria, sperm, and the like. Table 1provides a list of different examples of motile blood cells, theirtypical concentration in human blood, and an appropriate cross-sectionalarea of a rectangular shaped channel for allowing migration of themotile cells. Table 1 also lists channel cross-sectional areas for othermotile cells such as bacteria, parasites and sperm cells. The device canalso be used with cells from blood of other animals, e.g., murine,rabbit, monkey, or canine blood, as well. However, the dimensions of thepassageways 110 should be modified to accommodate the different sizes ofcells obtained from these other types of blood. For example, neutrophilsand RBCs from murine blood are smaller than those of humans (murine RBCare about 5-6 μm in diameter which humans are about 7 μm, and murineneutrophils cell diameter ranges 5-6 μm while human neutrophil celldiameter ranges 7-8 μm).

TABLE 1 Cells/μL blood - Typical channel size for healthy individualmigration Blood Cell Type Neutrophil (granulocyte) 5,000 6 × 6 μm²Eosinophil 10 6 × 6 μm² Monocyte 50 10 × 10 μm² Lymphocyte 3,000 8 × 8μm² Dendritic Cell 1 10 × 10 μm² Circulating endothelial cell 0.1 10 ×10 μm² Fibrocyte 0.1 6 × 6 μm² Mast cell 0.1 6 × 6 μm² Circulating tumorcell 0.01-1 10 × 10 μm² Platelets 50,000 2 × 2 μm² Other motile cells incomplex mixtures Bacteria 1 × 1 μm² Parasites (sporozoite phase) 5 × 5μm² Sperm cells 2 × 2 μm²

The movement of undesired cells relative to the movement of desiredmotile cells also can also be restricted by adding a relatively sharpturn in the passageway 110, e.g., a turn of at least about 90 degrees.Such a turn creates congestion/gridlock in the movement of undesiredcells. In particular, as a cell moves, tumbles, floats, or is pushedinto the corner, it tends to block the advance of other trailing cellsbehind it by restricting the cross-section of the channel to less thanthe diameter of a single cell. This configuration works well for cellsthat move based on granular flow (e.g., RBCs), because the granular flowforce pushing the cells in the channel is not enough to deform the cellsthrough the restricted section. However, since the cross-section of thechannel is larger than that of the undesired cell, gaps still exist forthe desired motile cells to pass through.

As an example, referring again to FIG. 1B, the passageway 110 iscomposed of two portions in fluid communication with each other, inwhich a direction of cell movement in a first portion 112 is angled withrespect to a direction of cell movement in the second portion 114. Thefirst portion 112 is in fluid communication with an output of the inputchamber 102, whereas the second portion 114 is in fluid communicationwith an input to the migration channel 108. Preferably, the anglebetween the directions of cell movement in the two portions is sharpenough to trap the undesired cells and prevent them from dispersing intothe migration channel 108. The angle can be measured from a positionwhere the second portion would otherwise be co-linear with the firstportion. For example, in the baffle 106, the angle between the directionof cell movement in the first portion 112 and the direction of cellmovement second portion 114 is about 90 degrees. Other angles also canbe used. For example, the angle can include, but is not limited to,about 45 degrees, about 55 degrees, about 65 degrees, about 75 degrees,about 85 degrees, 105 degrees, 115 degrees, 125 degrees, or 135 degrees.The angle should be at least about 45° to effectively restrict movementof the undesired cells. While the angle between the two portions of thepassageway 110 further inhibits the movement of the undesired cells, thedesired motile cells can continue to progress toward the attractantgradient by moving through gaps in the passageway 110 left by theundesired cells.

The baffle 106 can include a single passageway 110 or multiplepassageways 110, each of which is in fluid communication with the inputof the migration channel 108 and each of which is configured to inhibitmovement of undesired cells as described above. For example, as shown inFIG. 1B, the baffle 106 includes several passageways 110, with the firstportion 112 of each passageway 110 separated by a wall 116, creating acomb-like structure. The baffle 106 may include, but is not limited to,between 5 and 30 passageways, e.g., 20 passageways. As shown in FIG. 1B,the first portion 112 of each passageway 110 may be arranged in parallelwith a first portion 112 of an adjacent passageway 110. The secondportion 114 of each passageway 110 can be coupled together to form asingle horizontal channel. Although each passageway 110 is shown to havea 90 degree turn, different angles can be selected for each passagewayas discussed above, and the angles can all be the same or different. Thelengths (i.e., the distance along the direction of cell transport) forthe first portions 112 can be in the range of between about 10 to about100 microns. For example, the length can be between about 20 to about 80microns, between about 30 to about 70 microns, or between about 40 toabout 60 microns. Other lengths also are possible. The length of thehorizontal channel (formed by the second portions 114 of each passageway110) can be in the range of about 15 to about 500 microns. For example,the length of the horizontal channel can be between about 50 microns toabout 400 microns, between about 100 microns to about 300 microns, orbetween about 150 microns to about 250 microns. Other lengths also arepossible. The width of the walls 116 separating the first portion 112 ofeach passageway can range from about 5 microns to about 100 microns,e.g., 10 microns.

The device 100 also may include an exit channel 120, e.g., an open-endedchannel that exits the device and is open to the fluid media outside thedevice, in fluid communication with the migration channel 108 at a pointbeyond the baffle 106 and before the migration channel 108 enters theinlet of the attractant chamber 104. As in the migration channel, thefluid in the exit channel does not move or flow during the monitoring ofchemotaxis. The exit channel 120 creates a bifurcation 130 that allowsone to monitor the ability of motile cells to follow the attractantgradient toward the attractant chamber. For example, if one or moremotile cells migrate towards the exit channel instead of towards theattractant chamber, this may be an indication that the motile cell isdamaged or functioning improperly. Thus, the presence of the exitchannel allows one to quantify the number of desired cells thatcorrectly follow the attractant gradient, as opposed to moving into theattractant chamber. Alternatively, the migration of motile cells throughthe exit channel may be an indication that the attractant is inducingchemokinesis and not chemotaxis of the desired cell.

Both the exit channel 120 and the migration channel 108 should be sizedto allow at least the desired cells to pass through. For example, theheight of the exit channel 120 and/or the migration channel 108 can bebetween about 1-3 microns, though larger heights also can be used. Thelengths (distance along the direction of propagation) of the exitchannel 120 and the migration channel 108 can be between about 10-2000microns, e.g., 75 microns long. The widths of the exit channel 120 andthe migration channel 108 can be between about 8-12 microns, thoughother widths may also be used.

As shown in FIG. 1A, the device 100 of this embodiment includes multipleattractant chambers 104 in fluid communication with the input chamber102, although a device with a single attractant chamber can be used. Ifdesired, two or more of the attractant chambers 104 may be loaded withdifferent attractants. Accordingly, the effect of different attractantson the directionality and responsiveness of motile cells from a singlefluid sample can be studied simultaneously.

In some implementations, the baffle's ability to restrict the movementof undesired cells can be enhanced by adding antibodies to the surfacesenclosing the baffle's passageways. The antibodies can be selected tospecifically bind to RBCs (e.g., GlyA+) or other undesired cells (e.g.,CD14+ can be used for monocytes), further reducing the number of theundesired cells that pass into the migration channel. In addition, oralternatively, other agents may be added to the baffle surfacesenclosing the passageways. For example, the surfaces can be coated withagents, such as proteins, glycoproteins, or combinations of them. Suchcoatings can prevent the absorption of soluble factors to the surfaces,and facilitate migration of the desired motile cells and/or impede themovement of undesired cells. With certain motile cells, such asneutrophils, it can be important in certain embodiments not to includeany antibodies or other agents in the device that activate the motilecells in a way that can alter their motility.

Other areas of the device may also be coated with agents forfacilitating or modifying the motile cell functionality. For example, asan alternative or in addition to coating the baffle, the inlet chamberand/or migration channel can be coated with agents. The agents caninclude proteins such as albumin for preventing surface adhesion ofneutrophil. Alternatively, the protein can be configured to promoteneutrophil adhesion, such as P selectin.

In some implementations, a repellant can be used in the device toinfluence motile cell directionality. For example, a repellant may beadded to the exit channel or in a separate repellant chamber that is influid communication with the migration channel. Examples of repellantsinclude Slit2, Slit3, high concentrations [mM] of IL-8, DipeptidylPeptidase IV, and quorum sensing bacteria. Other chemorepellents areknown to affect different types of motile cells and can be selected bythose skilled in this field.

Device Fabrication and Assay Preparation and Use

As one example, the microfluidic device 100 can be manufactured usingthe following methods. First, a mold defining the features of the device100 is obtained. For example, the mold can be formed by applying andsequentially patterning two layers of photoresist (e.g., SU8, Microchem,Newton, Mass.) on a silicon wafer using two photolithography masksaccording to known methods. The masks can contain features that definethe different aspects of the device 100 such as the input chamber, thebaffle, the migration channel, the attractant chamber, and the exitchannel. The wafer with the patterned photoresist then may be used as amaster mold to form the microfluidic parts. A polydimethylsiloxane(PDMS) solution then is applied to the master mold and cured. Aftercuring, the PDMS layer solidifies and can be peeled off the master mold.The solidified PDMS layer includes grooves and/or recesses correspondingto the passageways, migration channels, exit channels, and attractantchamber of the device 100. In some implementations, the mold pattern isdesigned to include the features of multiple devices 100. Each devicecan be cut out from the PDMS layer using, for example, a hole puncher(e.g., a 5 mm hole puncher). Similarly, the input chamber also can beformed by using a smaller hole puncher (e.g., a 1.5 mm diameter holepuncher) to punch out PDMS material from the PDMS layer. The PDMSdevices then are bonded to a substrate such as a glass slide ormulti-well plate (i.e., each device is positioned in a correspondingwell of the well plate). For example, a bottom surface of the PDMSdevices can be plasma treated to enhance the bonding properties of thePDMS. The plasma treated PDMS devices then are placed on the glass slideor into the bottom of a well on a plate and heated to induce bonding.The microfluidic channels of the device can also be exposed to plasmatreatment prior to bonding to render the channels hydrophilic.Hydrophilic channels can enhance priming of the device with theattractant due to capillary wicking effects. FIG. 1A is a schematicdepicting a perspective view of a microfluidic device 100 fabricatedaccording to the foregoing procedures.

The example of a microfluidic device 100 described above, includes asubstrate layer of glass and a top layer of PDMS in which the inputchamber 102, the baffle 106, the migration channel 108 and theattractant chamber 104 are formed. In other implementations, both thesubstrate layer and the top layer can be PDMS substrates or othersimilar materials.

In general, the top layer (or the bottom layer) in which the baffle,migration channel, exit channel and attractant chamber are formed shouldbe selected to have the following characteristics. The layer can begas-permeable so that air in the baffle, migration channel, andattractant chamber can be displaced through the layer, either by pumpingfluid into the device or by placing the device under vacuum.Furthermore, the layer can be transparent so as to facilitate imagecapture of cell motility within the device. As explained above, thesurfaces of the baffle walls enclosing the passageways 110 may be coatedwith agents, for example, antibodies, to facilitate capture of undesiredcells from the fluid sample.

FIGS. 3A-3D are schematics depicting examples of the different stages inpreparing the device 100 for an assay. On the left side of each of FIGS.3A-3C, the microfluidic device is shown situated in a petri dish. Theright side of each of FIGS. 3A-3C depicts a cross-section of the inputchamber (labeled “whole blood reservoir”), the migration channel(labeled “neutrophil migration channel”), and the attractant chamber(labeled “chemokine reservoir”) of the microfluidic device. Though thefigures reference neutrophil migration channel and whole bloodreservoir, the process shown is applicable to other motile cells andfluid samples as well. As shown in FIG. 3A, a first stage of assaypreparation includes priming the device with an attractant solution (forexample, a chemokine solution, as referred to in FIG. 3A). Variousattractant solutions can be selected based on the motile cell to beanalyzed.

Examples of attractant solutions for neutrophils includeN-formyl-methionyl-leucyl-pheylalanine (fMLP), leukotriene B₄ (LTB₄),interleukin-8 (IL-8), the protein fragment C5a, adhenosine tri-phosphate(ATP), tumor growth factor beta (TGFb), or endothelial derivedneutrophil attractant factor (ENA), or the like. In someimplementations, the attractant solution includes the extracellularmatrix protein fibronectin to promote neutrophil surface adhesion.Preferably, the attractant solution is added shortly after performingplasma treatment and bonding the device so that the hydrophilic propertyof the microfluidic channels has not dissipated and can assist primingthe channels through capillary effects. Other methods of rendering theinner surfaces of the device hydrophilic can also be used. Othermaterials that are inherently hydrophilic can also be used tomanufacture the device. The attractant solution can be added to thedevice through the input chamber, e.g., through pipetting using agel-loading tip and surrounding the circumference of the whole device.

The device 100 then is placed under vacuum. By applying a vacuum to thedevice 100, the attractant solution is forced completely into thefluidic channels and all chambers of the device 100. At the same time,the vacuum causes any air present in the channels or chambers to diffusethrough the gas-permeable material of the top layer (e.g., the PDMSlayer). This process removes air bubbles that would otherwise be presentin the fluidic channels of the device, and which could potentially blockthe passage of cells through the baffle and migration channel. Toestablish the vacuum, the device can be placed into a desiccator, inwhich air pressure is reduced to a vacuum level of about 17-25 inches ofwater, for at least about 15 minutes.

In a second stage (FIG. 3B), the device 100 then is removed from thevacuum and the input chamber is washed to remove excess attractantsolution. For example, the input chamber can be washed using a phosphatebuffer saline (PBS) solution. The attractant solution in the baffle,migration channel, and attractant chamber remains in the device 100.After washing, the wells in which the devices 100 sit are filled with afluid suspension (referred to as “cell culture media” in FIG. 3B) thatis free of the attractant and allowed to sit for a specified time sothat a stable attractant gradient can form between the attractantchamber and the input chamber. The exit channel is surrounded by thefluid suspension and therefore acts as a sink for the attractant. Sincethe gradient in the exit channel decreases away from the migrationchannel, normal functioning cells will not migrate toward the exitchannel. The fluid suspension can include media solutions such as RPMI1640 media.

After establishing the attractant gradient, the fluid sample of interestis introduced into the input chamber 102 of the device in a third stage(FIG. 3C). For example, using gel-loading tips, samples of whole blood(or samples containing isolated motile cells) can be pipetted into thechamber 102. Once the fluid sample is in place in the chamber 102,static fluid pressure causes some of the cells of the sample to move,e.g., by granular flow (e.g., like grains of sand tumbling down anincline) into the passageways 110 of the baffle 106, where the desiredmotile cells begin migrating in a direction of the attractant gradient(see, e.g., FIG. 3D). Various properties of the motile cells may bemonitored in the device include, for example, the absolute number ofdesired motile cells that reach the attractant chamber, the number ofdesired motile cells that reach the attractant chamber relative to thetotal number of cells (motile and/or non-motile) in the fluid sample,the rate at which the motile cells reach the attractant chamber, and/orthe directionality of motile cells in the device 100.

In the case of whole blood, the RBCs will move according to a granularflow pattern. Once the RBCs reach the turns in the passageways 110, thecell movement will slow or stop and cause a backup of trailing cells. Incontrast, healthy neutrophils in the sample will continue to follow theattractant gradient and squeeze through openings left by the RBCs in thepassageways 110. The healthy neutrophils will then proceed through themigration channel 108 towards the attractant chamber 104. Unhealthyneutrophils may continue migrating into the exit channel 120 or neverreach the attractant chamber 104. During the migration assays, thedevice 100 can be maintained at a temperature suitable for cellmigration. For example, in the case of neutrophil migration, the device100 can be placed in a biochamber and heated to about 37° C. and havinga 5% CO₂ atmosphere with 80% humidity to maintain the viability of thecells. The humidified environmental chamber can, in certainimplementations, increase the observation duration several hours.

FIG. 4 is a schematic of an example of a system 400 for analyzing theproperties of motile cells following an attractant gradient, andincludes a microfluidic device 100. The microfluidic device 100 includesa baffle for inhibiting the movement of undesired cells in a fluidsample relative to the movement of desired cells, a migration channel,and an attractant chamber for establishing an attractant gradient. Thedevice 100 is held in a container 450, such as a petri dish or the like.The system 400 includes an imaging system 430 configured to captureimages and/or video of the cell migration. For example, the imagingsystem 430 is configured to perform time-lapse imaging. An example of animaging system suitable for use to record images of the cell migrationis the Nikon Eclipse Ti microscope with 10-20× magnification.

The total time required to record the movement of the motile cellstoward the attractant chamber depends on various factors, including thebaffle passageway length, the migration channel length, the attractantbeing used, and the attractant gradient itself. Since neutrophilstypically require about 5 minutes to begin migration in response to anattractant gradient, a minimum time necessary for monitoring theneutrophil motion with the system 400 is no less than approximately 10minutes (e.g., a distance of neutrophil migration of about 150-200 μmfor a neutrophil migration rate of 18 μm/min). Longer monitoring timesalso may be required, depending on the nature of the particular assaybeing conducted. For example, the time to monitor motile cell movement(e.g., neutrophil movement) may be on the order of 20 minutes, 30minutes, 40 minutes, 50 minutes, 1 hour, or longer. While time-lapseimaging is one particular way for characterizing the cell migration, onecould also count the number of cells that reach the attractant chamberat the end of the assay, without the aid of time-lapse imaging.

The system 400 further includes a computer system 435 that isoperatively coupled to the imaging system 430. The computer system 435can include a computer-readable storage medium (for example, a hard diskand the like) that stores computer program instructions executable bydata processing apparatus (for example, a computer system, a processor,and the like) to perform operations. The operations can includecontrolling the imaging system 430 to capture images of the migration ofcells through the device toward the attractant chamber. In addition, thecomputer system 435 can receive the captured images from the imagingsystem 430, and process the images to obtain various parameters, e.g.,one or more of a migration speed of motile cells in a channel, a numberof motile cells reaching the attractant chamber, and a directionality ofthe motile cells. Directionality of motile cells can be quantified bycounting the number of motile cells that follow the attractant gradientinto the attractant chamber, as opposed to the exit channel.

In some implementations, the computer system 435 is configured toexecute computer software applications that perform statistical analysisof the data captured by the imaging system 430. For example, thecomputer system 435 can be configured to perform multivariate analysisto determine correlations between neutrophil migration speed andclinical parameters. In some implementations, experiments tocharacterize the formation of gradients inside the device in the absenceof motile cells can be performed by replacing all or portions of theattractant solution (for example, the fMLP) with a fluorescent agent(e.g., fluorescein) of comparable molecular weight, and analyzing thedistribution and changes in fluorescence intensity from time-lapseimaging using the imaging system 430 and the computer system 435.

Applications

The microfluidic motility assays and methods described herein can beused in various applications. For example, the measurement of neutrophildirectionality is important in patients at high risk for infection wheredirectionality of neutrophils is known to be impaired, such as thosewith burn injuries or tissue trauma, patients undergoing chemotherapy,neonates in intensive care units, and/or diabetics. Impaired and/orover-stimulated neutrophils may migrate away from the site of the injuryand therefore cause injury to healthy tissues. The devices disclosedherein provide a platform to analyze neutrophil behavior to determinethe extent of damage to the neutrophils. For example, the devices can beused to determine the percentage of neutrophils that behave abnormally,as well as the particular type of abnormal behavior, such as failing tofollow an attractant gradient or changes in migration rate. The devicescan be used on samples of whole blood without requiring a separateisolation step for the neutrophils, thus reducing processing time.Additionally, the use of whole blood preserves the natural environmentfor neutrophils without inducing neutrophil activation. The devices canbe designed to handle small quantities of fluid sample, e.g., sampleshaving a volume of about 2 microliters, or about 1 microliter. Thedevices can be used with blood obtained from humans or animal subjects.Both reductions in processing time and reduced sample volumerequirements are advantageous for clinical applications, where it maynot be feasible to obtain larger amounts of sample fluid, e.g., ininfants or small mammals.

In some implementations, the devices can be used to analyze efficacy ofone or more medications on neutrophil activity. For example, amedication that affects, e.g., enhances, neutrophil motility can beadministered to a subject (e.g., a patient having a burn injury or othertissue trauma) to vary the motility of neutrophils within the subject.The medication can include one or more of several modulators ofneutrophil migration such as endogenous modulators (e.g., acetylcholine,interleukin-10 (IL-10), TNFalpha, interleukin-1 (IL-1), interleukin-6(IL-6)), resolvins, lipoxins, or exogenous modulators (e.g., curcumin,lysophosphatidylglycerol, or cholinergic drugs). Blood samples then canbe obtained from the patient once or periodically after administrationof the drug. Using the devices and systems described herein, neutrophilactivity can be analyzed to determine the drug's effect on neutrophilmotility. In some situations, neutrophils obtained from a subject can bestudied over a one week period, for example, at 48 hour intervals. Todetermine a long-term effect of an injury and treatment on the subject,the study period can be expanded to longer periods, e.g., six months, atregular intervals. With respect to neutrophils, an increase in the rateof migration observed over such time intervals may indicate woundhealing. If the rate of migration of the neutrophils does not suggestwound healing, then a treatment can be altered to administer a differentdrug.

The device 100 shown in FIG. 1A includes multiple attractant chambers104 coupled to a single input chamber 102. Accordingly, the device 100,or similar devices having multiple attractant chambers, can be loadedwith multiple attractants (e.g., a different attractant for eachattractant chamber) to establish different attractant gradients. Usingthe systems disclosed herein, one can then analyze the responsivity ofmotile cells to the different attractant gradients. One can quantify aperson's neutrophil functionality after injury or infection (or anyperturbation to the immune system) as well as to measure the efficacy ofpotential drugs or treatments.

Further characterization of neutrophil motility using the microfluidicdevices described herein can have important diagnostic implications notonly for burn patients, but also for patients afflicted by otherdiseases that compromise neutrophil functions. For example, the devicecan be applied to analyze neutrophil motility in pediatric patients toidentify patients who are at a higher risk for certain diseases. Intransplantations, the device can be used to analyze neutrophilmotilities to determine if there is a correlation between neutrophilmotility under medication and the occurrence of complications, forexample, infections and rejections. By determining a range of neutrophilmotilities that correlate to low infection and at whichimmuno-suppressant functions are not suppressed, it may be possible tovary the quantities of immuno suppressant medication that is beingadministered to patients.

In some implementations, the devices can be used to screen hundreds,1000s, 10,000s, or 100,000s of small molecules or other chemical agentsfor their effect on motile cell motility, e.g., on neutrophilchemotaxis. That is, the devices can be used to screen such compounds tosee which, if any, have an effect on cell motility, and the degree towhich motile cells are affected.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1—Device Modeling

To understand how a group of RBCs move through a baffle passageway, a2-D finite-element model was employed using the COMSOL Multiphysics®software program, which performed biophysical modeling ofchemoattractant diffusion in the device. The model was based on a strongsolid-fluid coupling, which allows the incorporation of deformable solidbodies (e.g., RBCs) in fluid-filled channels. The channel geometries andinitial RBC positions were inputted into a custom-built finite-elementsoftware package PAK45 and run on a desktop supercomputer consisting of32 cores (Supermicro Super Server: 4×Eight-Core Intel Xeon Processor2.70 GHz; 512 GB total memory). Using the model, the granular movementof RBCs through small channels was simulated. This movement is a resultof the mutual interaction of many RBCs in the whole blood loadingchamber, pushing the RBCs at the periphery to enter the connectedmicrochannels. To simulate this force and induce movement, we assumedthat the top-most RBC in the channel experiences an external force(equal to 1/(50 g), where g is gravitational acceleration constant. Thiswas equivalent to a stack of twelve RBCs with 5% higher density thanthat of media pushing one RBC into the channels) in the y-direction. Theother RBCs below the topmost RBC had no externally applied forces.

FIGS. 5A-5D are schematics depicting the different stages of RBCs movingthrough a channel from an input chamber and subsequently traversing a90° turn. Three different passageway widths (9, 12, and 14 μm) wereexamined. Each RBC was assumed to have a diameter of 7.5 m. FIG. 5Ashows the initial RBC configuration of 10 RBCs in the entrance segmentof a baffle passageway where the passageway width is 9 μm. The scale baron the left of FIG. 5A is a color scale corresponding to differentinternal stresses that may be experienced by the RBCs during migration.FIG. 5B depicts the final steady-state configuration inside the 9 μmchannel. FIG. 5B shows that as soon as one RBC is pushed into thecorner, it substantially blocks the advance of all RBCs behind it byrestricting the cross section of the passageway to less than one RBCdiameter. Only two RBCs have moved pass the corner. This strategy issuccessful because the force pushing the RBCs is not enough to deformthe cells through the corner of the passageway. FIG. 5C depicts thefinal steady-state configuration for a 12 μm channel after the sameinitial configuration shown in FIG. 5A. FIG. 5D depicts the finalsteady-state configuration for a 14 μm channel after the same initialconfiguration shown in FIG. 5A. The results in FIGS. 5C-5D demonstratethat reaching a stable configuration is less likely in larger channelswhen progressively more and more RBCs traverse the corner as the channelwidth increases.

Example 2—Comparison of Baffle to Straight Channel Design

To analyze the effectiveness of the baffle design containing restrictedpassageway cross-sections and turns for blocking RBCs versus that of astraight channel, one of each different device design was fabricated andtested with fMLP and LTB4 chemoattractants. The device containing thebaffle was also analyzed for its ability to allow neutrophil migration.

Device Fabrication

Each microfluidic device was designed with three main components:chemokine side chambers (200×200 μm), a central whole-blood loadingchamber, and migration channels that did or did not contain a RBC baffleregion. The device containing the baffle included 10 short microfluidicchannels (length ˜75 μm) connected horizontally through an approximately200-μm-long channel to create several 90° bending sections capable oftrapping the RBCs in order to prevent them from dispersing into the restof the migration channel. All migration channels were designed to be 12μm wide and 3 μm high.

The microfluidic devices were produced by replica moldingpolydimethylsiloxane (Sylgard 184, Elsworth Adhesives, Wilmington,Mass.) on a master wafer fabricated using standard photolithographictechnologies with Mylar photomasks (FineLine Imaging, Colorado Springs,Colo.). After curing for at least 3 hours in an oven set to 65° C., thePDMS layer covering the master was peeled off and holes were punched.First, the central loading chamber was punched using a 1.5 mm puncherand then a 5 mm puncher was used to cut out the entire donut-shapeddevice (Harris Uni-Core, Ted Pella Inc., Reading, Ca). A 12-well platewas then plasma treated along with the PDMS donut-shaped devices andbonded on a hot plate set to 85° C. for 10 minutes.

Whole Blood Handling

Capillary blood (50 μL) was collected by pricking a finger of healthyvolunteers. The blood was then pipetted into an eppendorf tubecontaining a mixed solution of HBSS media, heparin anti-coagulant (1.65USP/50 μL of blood), and Hoescht stain (10 μL, 32.4 μM). The eppendorftube was then incubated for 10 minutes at 37° C. and 5% CO₂ to allow forproper staining of the nuclei. Afterwards, 50 μL of the finger prickblood was pipetted into media containing the same Hoescht stainconcentration as previously described and incubated for 10 minutes at37° C. and 5% CO₂. Using a gel-loading tip, 2 uL of whole blood wasslowly pipetted into the central input chamber of the device.

Establishing Chemoattractant Gradient

For the devices in which neutrophil migration was to be observed, thedonut-shaped devices were filled with the chemoattractant solution ofN-formyl-methionyl-leucyl-phenylalanine (fMLP) [100 nM](Sigma-Aldrich,St. Louis, Mo.) or Leukotriene B4 (LTB4) (Caymen Chemicals, Ann Arbor,Mich.) [100 nM] immediately after the PDMS donut-shaped device wasbonded to the well plate. The chemoattractant solution also containedfibronectin [25 nM](Sigma-Aldrich, St. Louis, Mo.) to promote neutrophilsurface adhesion. The chemoattractant was pipetted into the whole bloodloading chamber (WBLC) and directly around the circumference of thedevice. The glass bottom 12-well plate was then placed in a desiccatorto de-gas for 15 minutes to ensure proper filling of the chambers whilethe PDMS surface was still hydrophilic from plasma treatment.Afterwards, the central whole-blood loading chamber and the outsideregion surrounding the donut were washed thoroughly with PhosphateBuffered Saline (PBS) in each well to wash away excess chemoattractant.The wells of the plate were then filled with RPMI 1640 media and allowedto sit for a period of 15 minutes to generate stable chemoattractantgradients.

Results

Time-lapse imaging was performed on a Nikon Eclipse Ti microscope with10-15× magnification and a biochamber heated to 37° C. with 5% CO₂ and80% humidity. For each experiment in which an attractant gradient wasestablished, at least 50 neutrophils were manually tracked.

FIG. 6A is a bright-field (BF) image of the device as fabricated abovewithout a baffle to filter the RBCs. RBCs are seen to clog the migrationchannel and contaminate the attractant chamber (see arrows). FIG. 6B isa BF image of the device as fabricated above with a baffle to filter theRBCs. The incorporation of the RBC baffle upstream of the migrationchannel significantly reduces RBC contamination in the attractantchamber by 63% and eliminates RBCs at the channel exit. Thus, the bafflecontaining the comb design proved more efficient in blocking theentrance of RBCs in the migration channels compared to the straightchannels.

FIGS. 7A-7H are time-lapse images of the device containing thecomb-shaped baffle, where a LTB₄ chemoattractant gradient wasestablished between the attractant reservoir and the input chamber. Asshown in FIGS. 7A-7H, few RBCs are able to enter the migration channel,while a neutrophil is able to migrate past the RBCs into the migrationchannel (see arrow in FIG. 7H). These results demonstrated that themicrofluidic device is capable of inhibiting the movement of the RBCsrelative to that of the neutrophils, without causing perturbations incell motility or directionality. Integration of the on-chip baffle inthe novel microfluidic device removes RBCs from actively migratingneutrophils and circumvents the need for cumbersome cell separationmethods such as density gradients, positive selection, or negativeselection, which are prone to introduce artifacts by activatingneutrophils. The results also show that the microfluidic device producesa stable linear chemoattractant gradient without the need forperipherals like an outside pressure source (i.e. syringe pump).

Example 3—Assay Validation with Finger Prick and Venous Healthy DonorBlood Sources

The microfluidic devices were also validated by loading whole blood fromfinger prick and venous sources, as well as isolated neutrophils towardthe chemoattractant fMLP. Specifically, neutrophil migration wasanalyzed for whole blood from the finger prick, from the venous bloodsource, and from the isolated neutrophils. The devices were fabricatedand the fMLP chemoattractant gradient was established as explained abovein Example 2.

Whole Blood Handling

Capillary blood (50 μL) was collected by pricking a finger of healthyvolunteers. The blood was then pipetted into an eppendorf tubecontaining a mixed solution of HBSS media, heparin anti-coagulant (1.65USP/50 μL of blood), and Hoescht stain (10 μL, 32.4 μM). The eppendorftube was then incubated for 10 minutes at 37° C. and 5% CO₂ to allow forproper staining of the nuclei. For venous blood samples, 10 mL ofperipheral blood was drawn from a health volunteer into tubes containing33 US Pherparin (Vacutainer, Becton Dickinson, Franklin Lakes, N.J.).Afterwards, 50 μL of the blood was pipetted into media containing thesame Hoescht stain concentration as previously described and incubatedfor 10 minutes at 37° C. and 5% CO₂. Using a gel-loading tip, 2 uL ofwhole blood was slowly pipetted into the central input chamber of thedevice.

Neutrophil Isolation

To compare the whole blood results with neutrophil migration from anisolated sample, we also isolated human neutrophils from whole bloodusing HetaSep followed by the EasySep Human Neutrophil Enrichment Kits(STEMCELL Technologies Inc. Vancouver, Canada) following themanufacturer's protocol. The final aliquots of neutrophils werere-suspended in 1×HBSS+0.2% human serum albumin (Sigma-Aldrich, St.Louis, Mo.) at a density of ˜40,000 cells/L and kept at 37° C. celluntil devices were properly primed.

Results

Time-lapse imaging was performed on a Nikon Eclipse Ti microscope with10-15× magnification and a biochamber heated to 37° C. with 5% CO2 and80% humidity. For each experiment in which an attractant gradient wasestablished, at least 50 neutrophils were manually tracked over a periodof 200 min. Directionality of primed neutrophils was quantified bycounting the number of cells that followed the chemotactic gradient andturned at the bifurcation toward the chemoattractant chamber as opposedto the number of cells that exited the device to the peripheral region.Cell velocities were calculated using Image J (NIH) and data analysiswith GraphPad Prism.

FIG. 8A is a plot of neutrophil migration counts among venous bloodsource, finger prick blood source, and isolated neutrophils. A similardelay time, accumulation rate and final cell count are observed in allthree conditions migrating to fMLP [100 nM]. Graphs correspond toaverage cell counts (n=16) in all attractant chambers. Neutrophils fromthe two whole blood sources, as well as isolated neutrophils, beganmigrating towards the fMLP [100 nM] gradient within 20 minutes andneutrophil accumulation numbers from all three sources were consistentaround 135±20 cells/device after 3.5 hours (P-value<0.001, R²=0.98). Therate of neutrophil accumulation remained constant for the length of the200 min experiment in all blood sources. As shown in FIG. 8A, weobserved higher variability of neutrophil counts with the finger prickblood source compared to the venous or isolated neutrophil sources,which may reflect higher heterogeneity of the neutrophil population inthe capillaries compared to the whole blood. These results suggest thatthe device will operate just as well as devices that require neutrophilisolation, but without the need to include the preliminary isolationstep.

We then measured variability between healthy donor neutrophil migrationfrom whole blood finger source towards fMLP [100 nM]. FIG. 8B is a plotof neutrophil migration counts per chamber compared for the 7 healthyvolunteers. Of interest, for 5 out of 7 donors' neutrophil migrationcounts clustered tightly around the average of 92±35 cells after 200min. However, two donors had significantly higher neutrophil migrationcounts, which may be representative of the variation in innate immuneresponse in the human population.

To determine device-device variation, we loaded whole blood from afinger prick of a healthy donor into 6 separate devices and quantifiedneutrophil accumulation to a gradient of fMLP [100 nM]. FIG. 8C is aplot of the average neutrophil migration counts for the fMLP gradient inthe 6 separate devices. The device-device variation was 14.1% (83±12cells after 200 min.), over the duration of the experiment.

We also established a healthy donor baseline, measuring neutrophilaccumulation from finger prick whole blood from the same healthy donorat one week intervals for a total of three weeks. FIG. 8D is a plot ofthe base-line for neutrophil migration in the healthy donor over thethree week time period. The experiment yielded equivalent neutrophilaccumulation values, thus suggesting high experimental reproducibilityas well as a consistent accumulation baseline for the same volunteer.The confirmation of a consistent baseline of neutrophil recruitment in ahealthy volunteer, suggests that perturbations in this baseline couldrepresent significant clinical changes in the innate immune response,such as injury, infection or dysfunction that would be useful indiagnosis or predictions of future clinical outcomes.

Example 4—Neutrophil Chemotaxis Towards Standard Chemoattractants

Neutrophil migration toward different attractants (fMLP and LTB₄) wasalso examined. The devices were fabricated and the fMLP and LTB4chemoattractant gradients were established as explained above in Example2. The concentrations of the three different chemoattractants werevaried from 10 nM to 50 nM to 100 nM. Solutions containing fibronectin[25 nM] and exclusive of fibronectin were prepared. Finger prick bloodand venous blood were obtained as described above in Example 3. Velocitymeasurements were obtained as explained above in Example 3 usingtime-lapse imaging. Neutrophil migration toward the fMLP and the LTB₄chemoattractants were compared against a control device, in which nochemoattractant gradient was established.

Results

FIG. 9A is a plot of the dose-response of neutrophils migrating out ofwhole blood to the LTB₄ and fMLP chemoattractants. The plots correspondto an average cell count reached in 16 different attractant chambers. Asshown in FIG. 9A, the neutrophils do not migrate in the absence of achemoattractant gradient. Overall, increased cells migration wasobserved at higher concentration. Maximal cell recruitment was observedat the 100 nM concentration in the attractant chamber of themicrofluidic device. As shown in FIG. 9A, a gradient of fMLP [100 nM]recruited neutrophils from finger pick whole blood at a two-fold highercount (86±7 cells/device after 200 min) than LTB₄ (39±12 cells/deviceafter 200 min). FIG. 9B is a plot of neutrophil velocity from fingerprick whole blood in response to fMLP and LTB₄. As shown in FIG. 9B,neutrophil velocity was comparable for both fMLP (19±6 μm/min) and LTB₄(20±7 μm/min). Neutrophil velocities were also consistent between venouswhole blood and finger prick whole blood for both fMLP and LTB4chemoattractants.

The bifurcation in the microfluidic device design (i.e., where the exitchannel splits off from the migration channel, see FIG. 1A) allows forthe quantification of neutrophils directionality by comparing the numberof cells that migrated towards the chemoattractant gradient to thenumber of cells that become “lost” and exit the device. This directionalindex is clinically relevant as it provides a quantitative measurementfor correct neutrophil response to a site of injury or infection. The“lost” or non-directional neutrophils would potentially migrate andcause unnecessary damage to healthy tissue or organs. FIG. 9C is a plotthat shows a directionality index for neutrophils in response to bothfMLP and LTB₄. Directionality is calculated by finding a ratio of thecells that correctly follow the attractant gradient to the attractantchamber divided by the total number of cells (cells that migrate to theattractant chamber plus the cells that exit the device). As shown inFIG. 9C, neutrophils from finger prick and venous healthy donor wholeblood sources have a directionality index greater than 0.9 for both fMLPand LTB₄.

The effect of fibronectin inclusion in the chemoattractant solution wasalso analyzed. Fibronectin promotes neutrophil adherence and acts as ablocking agent (in addition to 0.2% human serum albumin) for the glasssurface of the device. FIG. 9D is a plot that shows neutrophil count inthe fMLP attractant chamber for pin prick and venous blood in responseto attractant with and without fibronectin. As shown in FIG. 9D,fibronectin does not appear to change final neutrophil counts migratingtowards fMLP. The foregoing results demonstrate that the device issuitable for comparing motile cell response to different attractantgradients.

Example 5—Disfunction in Neutrophil Recruitment after Burn in HumanPatient

We also utilized the novel microfluidic device to monitor neutrophilchemotaxis function in a burn patient.

Blood Samples

Blood samples of 1 mL were collected from one burn patient sufferingfrom 24% total body surface area (TBSA) burn. Procedures for fabricatingthe device, preparing the chemoattractant gradient, and performingtime-lapse imaging were conducted as explained above with respect toexamples 2-4.

Results

Neutrophil chemotaxis was monitored over a 3 week treatment period. FIG.10A is a plot of neutrophil migration counts in the attractant chamberwith no chemoattractant gradient, with fMLP chemoattractant gradient[100 nM], and with LTB₄ chemoattractant gradient [100 nM]. FIG. 10B is aplot of velocity [μm/min] of neutrophils migrating to LTB₄ compared withfMLP over a three week period. FIG. 10C is a plot of directionalityindex of neutrophils migrating to LTB₄ compared with fMLP. All graphscorrespond to average cell counts across 16 attractant chambers.

As shown in FIG. 10A, there was an order of magnitude decrease inneutrophil cell count compared with the average range (shown with dottedline) of a healthy volunteer 1 week after the burn injury. Moreover, asshown in FIGS. 10B and 10C, we observed a 75% reduction in neutrophilvelocity and a 50% reduction in directionality in neutrophils migratingtoward a fMLP [100 nM] gradient. The number of cells accumulatingtowards fMLP spiked from below normal values to 15% above the normalhealthy volunteer range at two weeks post burn, which corresponded to aperiod when the patient was observed to have a fever. At two weekspost-burn, neutrophil velocity remained impaired in both fMLP and LTB₄conditions (60% and 40% reduction respectively), but neutrophildirectionality had been restored to the range of healthy volunteers.Three weeks post-burn, neutrophil cell counts to fMLP were lower thanthe average healthy volunteer count, whereas LTB4 accumulation countswere in the normal range. Velocity and directionality were both restoredto the normal range 3 weeks post-burn. These results demonstrate themicrofluidic device may be useful for monitoring variation in cellmotility of subjects over prolonged periods.

Example 6—Comparison of Human, Rat and Murine Neutrophils

Animal models of human disease differ in innate immune responses tostress, pathogens, or injury. Current technologies for measuringneutrophil phenotype prevent precise inter-species comparisons becausethey require the separation of neutrophils from blood usingspecies-specific protocols. For example, current neutrophil separationmethods, developed originally for human donors, require large volumes ofblood and are less suitable for mice due to their significantly lowercirculatory volume. Therefore, many studies on mouse neutrophils aredone with bone marrow cells. However, bone marrow neutrophils appear tobe heterogeneous and functionally immature. Furthermore, standardnegative enrichment of neutrophils include a lengthy (3 hour) protocolsduring which the neutrophil phenotype can change. Moreover, antibodycocktails for neutrophil isolation are less specific for mouse thanhuman and activation levels of neutrophils affect the purity and yield.However, by using the novel microfluidic device described herein, weperformed a robust characterization of neutrophil migratory phenotypesfrom different species directly from a droplet of whole blood. Inparticular, using the new device, neutrophil measurements were performedfrom minute volumes (less than 2 μL) of whole blood (WB), from variousspecies donors (rat, murine and human), with high precision and singlecell resolution.

Microfluidic Device Fabrication

The microfluidic device to study mouse, rat and human neutrophilchemotaxis from one droplet of whole blood was designed with three maincomponents: focal chemoattractant chambers (FCCs) (200×200 μm), acentral whole-blood loading chamber, and migration channels containingRBC filtering regions. The filter for each migration channel included 10short channels (Length ˜75 μm) with a 3.5 μm narrowing region (‘pinch’)connected horizontally through an approximately 200-μm-long channel tocreate 90° bending sections capable of trapping the RBCs to prevent themfrom dispersing into the rest of the migration channel. All migrationchannels were designed to be 12 μm wide and 3 μm high to establish onlya single column of RBCs for efficient trapping while allowing activemouse, rat and human neutrophils to easily migrate through. The deviceswere fabricated as described above in Example 2.

Whole Blood Sample Collection

For humans, 50 μL of capillary blood was collected by pricking a fingerof healthy volunteers. For mice, 50 μL of capillary blood was collectedby the facial vein method (Institutional animal care and use Protocol#2007N000136) requiring no anesthesia. For rats, 50 μL of venous bloodwas collected from the tail vein (Institutional animal care and useProtocol #2012N000034) using 1-2% Isoflurane inhalant. The blood wasthen pipetted into an eppendorf tube containing a mixed solution of HBSSmedia, heparin anti-coagulant (1.65 USP/50 μL of blood), and Hoechststain (10 μL, 32.4 μM). The eppendorf tube was then incubated for 10minutes at 37° C. and 5% CO2 to allow for proper staining of the nuclei.

Device Priming and Cell Loading

All reagents and whole blood were pipetted into the device and there wasno flow or requirement of external syringe pump. A gradient of thechemoattractant was established along the migration channels bydiffusion between the chemoattractant chambers and the central loadingchamber. Prior to cell loading and immediately after bonding to the wellplate, donut-shaped devices were filled with the chemoattractantsolution containing 25 nM of fibronectin (Sigma-Aldrich, St. Louis,Mo.). The well plate was then placed in a desiccator under vacuum tode-gas for 15 minutes to ensure proper filling of the chambers while thePDMS surface was still hydrophilic. Afterwards, the central whole-bloodloading chamber and the outside region surrounding the donut were washedthoroughly in each well to establish the gradient along the migrationchannels. The wells of the plate were then filled with RPMI 1640 mediaand allowed to sit for a period of 15 minutes to generate stablechemoattractant gradients. Finally, using a gel-lading tip, 2 μL ofwhole blood was slowly pipetted into the central whole-blood loadingchamber.

Chemotaxis Imaging and Measurements

Time-lapse imaging was performed on a Nikon Eclipse Ti microscope with10-15× magnification and a biochamber heated to 37° C. with 5% CO2 and80% humidity. Separate experiments to characterize the formation ofgradients along the migration channels in the absence of cells wereperformed under similar temperature and gas conditions but by replacingthe chemoattractant with fluorescein (Sigma-Aldrich, St. Louis, Mo.) ofmolecular weight comparable to that of fMLP (MW=438) and LTB4 (MW=336).For each experiment, at least 50 neutrophils were manually tracked.Directionality of primed neutrophils was quantified by counting thenumber of cells that followed the chemotactic gradient and turned at thebifurcation toward the chemoattractant chamber as opposed to the numberof cells that exited the device to the peripheral region. Cellvelocities were calculated using ImageJ (NIH). Total percentage ofneutrophils to migrate was estimated using a COMSOL simulation modelthat estimated that 30.6% of area in whole blood loading chamber fromwhich neutrophils could migrate in experimental time to be abovecritical gradient concentration. For an average human experiment, thiswould estimate ˜277 neutrophils per well that are exposed tochemoattractant gradient.

Results

An important feature that enabled the use of WB directly in themicrofluidic device was the red blood cell (RBC) filter. Murine RBCs(average diameter=6 μm, thickness=1 μm) are of smaller geometry thanhuman (average diameter=7-8 μm, thickness=2 μm). The RBC filter combinedflat channels, a comb of 90° angles, and a 3.5 μm ‘pinch’ of squarecross section to prevent the granular-flow of RBCs into the neutrophilmigration channels. RBCs pushing on each other under the effect ofgravity, were mechanically blocked at the entrance of the channels andremained confined inside the central loading chamber. The neutrophilmigration channels remain clear because the blocked RBCs do not clog thechannel and sufficient space remains between the RBC membrane and thechannels walls to allow chemokine diffusion. Thus, the formation ofneutrophil-guiding gradients from the WB to the FCC, along the migrationchannel, was unperturbed. Neutrophils were able to actively deform andmigrate through the pinch, which assured the selectivity of the assay bypreventing the migration of lymphocytes and monocytes, which deform lessand require larger channels for migration. The selectivity was verifiedby observing the characteristic polymorph shape of the nucleus of movingcells. Once the neutrophils passed the pinch, they continued to followthe chemoattractant gradient along the migration channel and enter theFCC. The bifurcation in the channel created a ‘decision point’ whereneutrophils can migrate toward or away from the chemoattractantgradient, providing critical information about their directionality.

To compare neutrophil migration phenotype between species, we measuredchemotaxis to two standard chemoattractants (fMLP and LTB₄) in humans,C57BL/6 and Sv129S6 mice and Wistar rats. Human neutrophils in 2 μLwhole blood samples migrated towards fMLP (55.8±19.8%) and LTB4(54.0±5.6%) (see FIG. 11A). A lower percentage of Wistar rat neutrophilsmigrated to fMLP and LTB₄ (10.9±8.5 and 2.8±1.1%, respectively).Surprisingly, LTB₄ was the only chemoattractant able to inducesignificant neutrophil migration in all mouse neutrophils tested:Sv129S6 (99.5±13%) and C57BL/6 (52.7±24%). The velocity of C57BL/6neutrophils towards fMLP (13±7.4 μm/min) was ˜2.5-fold lower than thatof Sv129S6 (19.7±8.7 μm/min), rat (26.2±5.9 μm/min), or human (23.4±5.4μm/min) neutrophils (see FIG. 11B). The directional index of C57BL/6neutrophils toward fMLP (0.61±0.07) was comparable to that of ratneutrophils (0.62±0.12) and significantly lower than that of Sv129S6(0.82±0.06) or human (0.85±0.06) neutrophils (see FIG. 11C). Thevelocity and directionality deficits in C57BL/6 neutrophils havemultiplicative effects and suggest less effective neutrophil migrationupon stimulation.

The directionality of Sv129S6 mouse neutrophils towards C5a was lowerthan in humans, while directionality towards LTB₄ was comparable (FIGS.12C-D). C5a activation of mouse neutrophils led to random migration.These patterns of neutrophil migration in response to C5a are consistentwith migration patterns reported previously from isolated humanneutrophils. More human neutrophils migrated in response to C5a in theFCC than mouse neutrophils (26.8±5.9% versus 1±0.8%, FIG. 12A), and moreneutrophils were activated by C5a compared to Sv129S6 mouse neutrophils(63.3±7.6% versus 18.3±3%, FIG. 12B).

The results demonstrate fundamental differences in neutrophil migratoryresponses between mice, rats and humans. Amongst hundreds of laboratorymouse strains available, two-thirds of all murine research is undertakenwith the C57BL/6J (B6) strain (compared to 1% for Sv129S6) because ofits robustness and availability of congenic strains. As therapies forhuman diseases become specifically targeted, it is increasinglyimportant to further understand mouse strain differences in innateimmune function and to wisely choose a mouse strain that most accuratelymodels the human response to disease or drug therapeutic interventions.Using the new microfluidic device described herein, our results showthat strain differences in neutrophil migratory function between commonlaboratory mouse models are significant and must be considered whenselecting the appropriate model to mimic human infection orinflammation.

The novel device and techniques described herein were used to allowprecise measurement of neutrophil chemotaxis from micro-volume samplesof murine, rat and human blood in the same conditions and following thesame sample preparation protocols. The assay was performed in thepresence of all blood components, and was highly multiplexed. Theresults demonstrate that the novel device and techniques may be used byresearchers to understand species and mouse strain differences inneutrophil migratory phenotypes from conscious animals over time.Compared to traditional methods (e.g., transwell assay), the deviceavoids lengthy neutrophil isolation steps, uses micro-volume amounts ofblood from conscious animals, which allows for repeated measures withoutpotential confounding effects of anesthetic drugs, and providessingle-cell-resolution information regarding neutrophil directionalityand speed. The novel microfluidic device and techniques described hereinalso have two specific advantages compared to recent techniques thatrely on neutrophil capture from blood by selective adhesion e.g.P-selectin. First, by avoiding the cell washing steps, the new devicepreserves the integrity of the blood sample, and with it important cuesthat may modulate neutrophil activity from serum or other cells in thewhole blood. Second, by relying on physical (channel geometry) ratherthan biological mechanisms (selectins or endothelial cells) to achieveselectivity for neutrophils, the device eliminates the artificialactivation of neutrophils via capture mechanisms and the need forspecies-matched capture molecules. The requirement of small numbers ofcells is particularly advantageous when studying mice, where bloodvolumes are limited. The novel device and techniques described hereinalso eliminate the necessity of pooling blood from several animals andpermits repeated single animal neutrophil phenotype data measurementsover-time, so as to potentially monitor progression of disease and/ortherapeutic responses. Measuring neutrophil migration in the whole bloodnative microenvironment mimics the in vivo, holistic animal responsemore accurately.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for monitoring neutrophil chemotaxis ina device, the method comprising: obtaining a device that comprises aninput chamber, an attractant chamber, a migration channel arranged influid communication between an outlet of the input chamber and an inletof the attractant chamber, a comb-shaped baffle arranged in fluidcommunication between the outlet of the input chamber and the migrationchannel or within the migration channel, wherein the baffle comprises afirst fluid passage in fluid communication with a second fluid passage,wherein an angle between a sample transport path in the first fluidpassage and a sample transport path in the second fluid passage is about90 degrees, and an exit channel in fluid communication with themigration channel at a point beyond the baffle and before the migrationchannel enters the inlet of the attractant chamber; adding an attractantsolution to the device to establish an attractant gradient between theinput chamber and the attractant chamber; adding to the input chamber ablood sample comprising a plurality of red blood cells and a pluralityof neutrophils; incubating the device under conditions and for a timesufficient to enable movement of cells in the blood sample from theinput chamber into the migration channel, wherein the baffle isconfigured to inhibit movement of the red blood cells through the baffleto a greater extent than the baffle inhibits movement of the neutrophilsthrough the baffle; and monitoring whether any of the neutrophils followthe attractant gradient in the migration channel toward the attractantchamber.
 2. The method of claim 1, wherein monitoring whether any of theneutrophils follow the attractant gradient comprises determining anumber of neutrophils that follow the attractant gradient.
 3. The methodof claim 1, wherein establishing the attractant gradient comprises:adding the attractant solution to all chambers and channels in thedevice; and replacing the attractant solution in the input chamber witha liquid medium that lacks the attractant such that the attractantgradient forms between the input chamber and the attractant chamber. 4.The method of claim 1, wherein a cross-sectional area of the first fluidpassage normal to the sample transport path in the first fluid passageis greater than a red blood cell cross-sectional area, and wherein aheight of the cross-sectional area is greater than a red blood cellthickness and less than a red blood cell diameter, and a width of thecross-sectional area is greater than the red blood cell diameter.
 5. Themethod of claim 1, wherein the baffle comprises a plurality of firstfluid passages, and a plurality of second fluid passages, each firstfluid passage being in fluid communication with the outlet of the inputchamber and in fluid communication with a corresponding second fluidpassage, wherein an angle between a fluid transport path of each firstfluid passage and a sample transport path of the corresponding secondfluid passage is greater than or equal to about 45 degrees.
 6. Themethod of claim 1, further comprising analyzing the health of theneutrophils that follow the attractant gradient in the migration channeltoward the attractant chamber.
 7. The method of claim 6, whereinanalyzing the health of the neutrophils comprises determining a numberof the neutrophils that follow the attractant gradient toward theattractant chamber compared to a total number of cells moving throughthe migration channel, determining a rate at which one or moreneutrophils follow the attractant gradient toward the attractantchamber, and/or determining a number of neutrophils that do not followthe attractant gradient compared to the total number of moving cells. 8.The method of claim 1, wherein monitoring whether any of the neutrophilsfollow the attractant gradient comprises obtaining an image ofneutrophils in the attractant chamber.
 9. The method of claim 1, whereinadding the attractant solution to establish the attractant gradientcomprises applying a vacuum to the input chamber, the migration channel,the baffle and the attractant chamber such that air is absorbed throughwalls of the device.
 10. A method of screening neutrophil attractants,the method comprising: obtaining a device that comprises an inputchamber, a plurality of attractant chambers, and a plurality offiltration passageways, each filtration passageway being in fluidcommunication with the input chamber and a corresponding attractantchamber, and each filtration chamber comprising a migration channel influid communication between an outlet of the input chamber and an inletof the corresponding attractant chamber, a comb-shaped baffle arrangedin fluid communication between the outlet of the input chamber and themigration channel or within the migration channel, and an exit channelin fluid communication with the migration channel at a point beyond thebaffle and before the migration channel enters the inlet of thecorresponding attractant chamber, wherein the baffle comprises a firstfluid passage in fluid communication with a second fluid passage,wherein an angle between a sample transport path in the first fluidpassage and a sample transport path in the second fluid passage is about90 degrees; adding a different attractant solution to at least twoattractant chambers to establish a different attractant gradient betweenthe input chamber and each attractant chamber to which an attractantsolution has been added; adding to the input chamber a blood samplecomprising a plurality of red blood cells and a plurality ofneutrophils; incubating the device under conditions and for a timesufficient to enable movement of cells in the blood sample from theinput chamber into one or more filtration passageways, wherein thebaffles of the one or more filtration passageways are configured toinhibit movement of the red blood cells through the baffles to a greaterextent than the baffles inhibit movement of the neutrophils through thebaffles; and monitoring whether any of the neutrophils follow any of theestablished attractant gradients to one of the attractant chambers towhich an attractant solution has been added.
 11. The method of claim 10,wherein monitoring whether any of the neutrophils follow any of theestablished attractant gradients comprises: for each differentattractant gradient, determining a number of neutrophils that follow theattractant gradient and/or determining a rate at which one or moreneutrophils follow the attractant gradient.
 12. The method of claim 10,further comprising identifying the attractant that establishes theattractant gradient resulting in the largest number of neutrophilsreaching an attractant chamber and/or resulting in the highest rate atwhich one or more neutrophils follow the attractant gradient.
 13. Themethod of claim 10, wherein a cross-sectional area of each first fluidpassage is greater than a red blood cell cross-sectional area, andwherein a height of the cross-sectional area for each first fluidpassage is greater than a red blood cell thickness and less than a redblood cell diameter, and a width of the cross-sectional area is greaterthan the red blood cell diameter.